Nexcelom Bioscience
  • Cellometer

Introduction to Stem Cells

The use of primary stem cells has become common place in fields such as cancer therapy, regenerative medicine, transplantation and basic oncology research. Although they are widely used, the generated stem cells are a precious commodity in any lab. Because stem cells, whether mesenchymal, embryonic or iPSC, are typically used in downstream experiments examining surface marker expression, cell differentiation and determining therapeutic potential the initial seeding density, as well as cell sample size, plays an important role in the use of these cells.

The Cellometer line of automated cell counters incorporates image based cell counting and fluorescence detection in a compact and easy-to-use instrument that reliably automates cell counting, viability determination and other fluorescent assays. By simply pipetting 20µL of sample into a disposable chamber, Cellometer acquires cell images that are analyzed to determine cell counts, concentration, cell sizes, and fluorescence intensity, typically in 30 seconds or less, making them an ideal instrument for low volume samples. Similarly, 20 µL is needed to perform GFP trasduction/transfection efficiencies, cell cycle analysis or any other cell-based-assay.

This combination of features makes it ideal for use in stem cell research. Samples can be counted and viability determined with Trypan Blue using the bright field Cellometer cell counters. Our fluorescent Cellometer cell counters and image cytometer systems combine this bright field functionality with fluorescent detection to also determine stem cell viability using fluorescent stains such as Propidium Iodide, and being able to accurately determine concentration of nucleated cells in human bone marrow or cord blood. The fluorescence imaging mode can also be used to quantify GFP transfection efficiency when transfecting stem cells.

Accurate Counting of Stem Cells

Cell images acquired in the brightfield mode are analyzed to determine total cell count. Concentration is automatically calculated, and all data is automatically saved or exported. This eliminates user to user variability and improves throughput over manual counting methods, especially when analyzing stem cells which can be clumpy or irregularly shaped.

Embryonic Stem Cells

Embryonic Stem Cells Embryonic Stem Cells counted

Figure 1: Images of embryonic stem (ES) cells acquired by Cellometer. Green circles indicate cells that have been counted by the Cellometer software.

Irregular & Clustered Stem Cells

Cell Clusters Cell Clusters counted

Figure 2: Cell clusters or irregular shaped cells can be accurately counted. Green circles indicate cells that have been counted by the Cellometer software.

Automated Determination of Stem Cell Viability

Stem cell viability can be determined by several methods. Dead cells stained with trypan blue can be analyzed in the brightfield mode (figure 3). Detecting viability via fluorescent dyes, such as propidium iodide (PI) can be performed by acquiring brightfield images for total cell concentration and fluorescent images to count PI stained dead cells.

Human Mesenchymal Stem Cells

Human Mesenchymal Stem Cells Human Mesenchymal Stem Cells counted

Figure 3: Analyzed brightfield images of Human mesenchymal stem cells (HMSC) cells show live cells circled in green and trypan blue stained dead cells circled in red.

Mouse Embryonic Feeder Cells

Bright field Mouse Embryonic Feeder cells PI stained Mouse Embryonic Feeder cells

Figure 4. Brightfield image of Mouse Embryonic Feeder (MEF) cells in the brightfield image, PI stained dead cells are visible in the fluorescence imaging mode.

Bone Marrow Stem Cells - CD34+

Bone Marrow Stem Cells - CD34+

Figure 5: Brightfield image of bone Mmarrow stem cells (left), AO/PI stained stem cells shown in the fluorescent image (right).

Accurate Counting of Bone Marrow and Cord Blood

Samples containing stem cells have large amounts of red blood cells (RBCs) or platelets present and can be difficult to count via manual methods. Because nucleated cells cannot be visually identified amongst these other cells, a fluorescent dye such as Acridine Orange (AO) can be used to positively identify nucleated cells.

Human Bone Marrow

Total Number Nucleated Cells in Bone Marrow can be Measured Using AO

Figure 6: AO stained nucleated cells in human bone marrow are identified and counted in the fluorescence image (right).

Cord Blood Samples

Total Number Nucleated Cells in Cord Blood can be Measured Using AO

Figure 7: Cord blood samples stained with AO/PI positively identifies live AO positive nucleated cells (in green), and dead nucleated cells (in Red) without the need for RBC lysis

Quantifying GFP Transfection Efficiency

The unique bright field/fluorescent imaging capabilities can also be used to quantify transfection efficiency when using reporter proteins such as GFP, without having to use complex methods, such as flow cytometry. A total count is acquired in the bright field mode, and GFP positive cells are counted in fluorescent mode to calculate the percentage of cells that are expressing GFP.

Transfected Mouse Embryonic Feeder Cells

brightfield image Mouse embryonic feeder cells fluorescent image Mouse embryonic feeder cells

Figure 8: Mouse embryonic feeder (MEF) cells that have undergone transfection. Total cell count is determined in the bright field image (left), GFP positive cells are indicated by green circles in the fluorescent image (right).

Example Results of Stem Cells Analyzed on Cellometer

Cell Type
Cell Concentration
Cell Detection/Viability
CD 34 +
4.81 x 106/ml
PI viability: 74.3%
Mouse embryonic feeder cell (MEF)
5.18 x 105/ml
AO or AO/PI
Mouse embryonic feeder cell (MEF)
2.48 x 106/ml
Trypan blue viability: 92.1%
Mouse embryonic feeder cell (MEF)
1.06 x 106/ml
PI viability: 91.0%
Transfected Mouse embryonic feeder cell (MEF)
Total: 3.52 x 106/ml
GFP: 8.24 x 105/ml
GFP
Human mesenchymal stem cells (HMSC)
6.49 x 106/ml
Trypan blue viability: 91.4%
Induced pluripotent stem cell (IPS)
7.51 x 105/ml
Trypan blue viability: 37.9%
Human cord blood
1.40 x 107/ml
AO or AO/PI
Human bone marrow
7.32 x 107/ml
AO or AO/PI
Adult cardiac stem cell
3.30 x 105/ml
Trypan blue viability: 85.3%
Human embryonic stem cell (HES)
2.38 x 106/ml
None
Transfected human embryonic stem cell (HES)
1.23x 106/ml
RFP: 8.32x105/ml
RFP
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